Intestinal glucose absorption of rat exposed to Curcuma longa (Yellow ginger) and Zingiber officinale (Common Ginger)

Andrea Carla M. AdajarNelle Rey Anne G. AsorShaira B. HernandezJanellaMariz N. JazarenoNosAmev V. Serrano

Group 3 Sec. AB-2L

May 25, 2015

ABSTRACTAn experiment on the effect of crude extracts of two ginger varieties, Zingiber officinale (common ginger) and Curcuma longa (yellow ginger), on the intestinal glucose absorption of rats was conducted. Results showed that both the ginger treated groups have a significant (=0.05) lower rate of glucose transport compared to the control group. Furthermore statistical analysis showed that rate of glucose transport is lower in C. longa (0.67mmol/L/min) than in Z. officinale treated rats (2.00mmol/L/min). The results led to the conclusion that C. longa is a more effective inhibitor of glucose transport than Z. officinale.

INTRODUCTION

Ginger has been an indispensable crop in Southeast Asia.In the Philippines, common ginger (Zingiber officinale) and yellow ginger (Curcuma longa) are widely cultivated. Both varieties have long been used in cooking and in medicinal purposes, specifically in treating wounds, diarrhea, rheumatism, and sore throat.Recent studies have shown that ginger has anti- microbial, anti- inflammatory, and possible anti- cancer properties (Habib et al., 2008). Ginger is a native plant in tropical Asia and is widely cultivated in the Philippines. It is used as a common ingredient in local dishes and brewed to produce medicinal tea called salabat.Aside from ginger, many past studies had been done to test the inhibitory effect of various plants on the transport of glucose. These researches also include the investigation of the compounds in the plant that cause such inhibitory effect. For example, Shirosaki, Goto, Hirooka, Masuda, Koyama, and Nazawa (2012) discovered that multiflorin from peach leaf extract is a strong inhibitor of glucose transporters in the small intestine of mice. However, a more related study conducted by Song et al. (2002) claim that the common ginger inhibits intestinal glucose transporters. Moreover, ginger was shown to normalize blood glucose levels.The specific objectives of the study are:1)to evaluate the inhibitory effect of the extract of two ginger varieties, namely, Curcuma longa and Zingiber officinale, on glucose absorption in rat small intestine; and2)to compare the effectiveness in the inhibition of glucose absorption of the two ginger varieties using one way ANOVA and Pairwise Mean Comparison Test.The study was conducted at the Animal House of the Institute of Biological Sciences from March 19 until April 19 and at room 405 of the New College of Arts and Sciences (NCAS) in the University of the Philippines Los Baos on April 20, 2015.

MATERIALS AND METHODS

Extraction of gingerFresh ginger rhizomes of Zingiber officinale(common ginger) and Curcuma longa (yellow ginger), which weighed approximately one kilogram each were purchased in a public market near the vicinity of the University. These were washed several times with water until the remnants of soil were removed. The two ginger varieties were separately peeled, sliced, and ground in a blender. The resulting extracts were then strained through a cheese cloth.Preparation of ratsFifteen male rats of approximately 200 grams were obtained from the Bureau of Food and Drugs, Alabang. The rats were then transported, and eventually were randomly grouped into three ,and were housed in separate cages made of chicken wire. The rats were acclimatized for one week; they were given normal rat food (i.e. pigeon pellets) at 20% of their body weight and were provided with ad libitum water. This was based on Zninorova, Lekhanya, Erlwanger, and Chivandis experimental methods in 2014.Experimental treatment set-upThe rats were randomly divided into three groups (n=5)based on the dietary treatment to be given control, common, and yellow. For the control group, ad libitum water was given. For the common and yellow groups, Zingiber officinale and Curcuma longa extracts were introduced, respectively. The amount of ginger extract fed, via gavage method, to each rat was based on a 4 mL per kilogram body weight. In addition to the extracts and ad libitum water, normal rat food was also given to each rat that was 10% of their body weight. This was based on the study conducted by Elshater, Salmann, and Moussa in 2009. Determination of intestinal glucose absorptionThe rats were fasted overnight; only water was provided. After fasting, the rats were sacrificed by anaesthetizing the rats using cotton soaked in chloroform. The method used in determining glucose absorption in small intestine was derived from Tuttle, Schottelius, and Thomsons Manual (1963). The abdominal cavity was dissected by carefully cutting through the abdominal wall. The surrounding mesenteries and fats were removed. Measuring from the duodenum, 18 cm of the small intestine was obtained by cutting it from the stomach and large intestine. The inside of the small intestine was flushed with Krebs PO4-Ringers solution until clear fluid flowed at the other end. It was then immersed in a Petri dish containing the Ringers solution. Afterwards, 4 cm of the small intestine was cut and tied at one end. The open end of the intestine was securely ties after placing the tip of the cannula inside it. In a 15 mL test tube, 10 mL of Krebs PO4-Ringers solution + glucose (500 mg glucose/100 mL Krebs PO4-Ringers solution) was placed. A stopper, containing the cannula, gut sac, and hypodermic needles was inserted into the tube upon submerging the gut into the Ringers + glucose solution. The inside of the sac was then filled with the Ringers + glucose solution up to about two cm above the tube fluid level. A fine stream of oxygen gas bubbles was then introduced into the fluid via the rubber tubing from the cylinder reducing valve, which was connected to the no. 21 hypodermic needle. The test tube set-up was immersed in a water bath at 37 C for 30 minutes. The set-up used was shown in Figure 1.

Figure 1. Set-up used in the determination of amount of glucose absorbed in the rat small intestine.

The initial glucose concentration of each set-up (concentration of the Krebs PO4-Ringers solution + glucose) was determined using Insight urinalysis strips. One drop of the solution from the intestine was placed on the strip; color changes were observed and were compared with a standard chart. The concentration was then recorded. Thirty minutes upon submersion and introduction of oxygen gas, the final glucose concentration was determined. Another drop of the solution inside the intestine was placed on the strip. The same procedure was done in determining the concentration. All the initial and final concentrations gathered were recorded and were tabulated.

Statistical analysisDifferences between obtained results were evaluated by methods of variation statistics using a parametric F-test using one way analysis of variance (ANOVA) for independent samples and were considered significant at = 0.05. Pairwise mean comparisons test was used to compare the means of ginger-treated groups with the control group.RESULTS AND DISCUSSION

The list of the final concentrations obtained in each set-up can be found in Table 1, where the average final concentrations of each treatment and the differences between the initial and the average final concentration are also listed.The initial glucose concentration represents the glucose concentration inside the intestinal gut before the occurrence of transport while the final glucose concentration represents the concentration inside the gut after transport. And the difference represents the overall rate of the transport of glucose. Since the set-ups were all observed within thirty minutes, a low mean difference corresponds to a low rate of glucose transport and the opposite is true for a high mean difference.

Table 1. Initial glucose concentration, average final glucose concentration, and average difference of the three set-ups.

Ginger varietyRat numberInitial glucose concentration (mmol/L)Final glucose concentration (mmol/L)Initial - Final

Yellow1110**

23080

31595

43080

53080

Total105335

Average26.2583.75

Control16050

21100

31100

46050

51100

Total450100

Average9020

Common1**

26050

33080

46050

5**

Total150180

Average5060

To be able to understand the results clearly, listed in Table 2 are the average rates of glucose transport computed. It can be observed that the control set-up has the highest rate of glucose transport, which means that it is the least inhibited, followed by those treated with common ginger and yellow ginger, respectively. The low glucose transport rate of the yellow ginger means that it is more inhibited than common and the control.

Table 2.Average rate of glucose transport of the ginger varieties.

Ginger varietyAverage rate of glucose transport (mmol/L/min)

Control2.79

Yellow0.67

Common2.00

The decrease in the rate of the glucose transport of the two ginger varieties corresponds to the results of Nwachuckwu and Ohiris study, which was conducted in 2012, where a decrease in the small intestinal blood glucose profile of the ginger-treated mice were observed after a period of treatment. They accounted their results to the effect of the ginger in bile production in the liver. They explained that ginger increases bile production and therefore, increases the rate of digestion, which causes food to move faster along the digestive tract and therefore, allow little time for glucose to be absorbed.

The results also correspond to the anti-hyperglycaemic effect of ginger treatment to Alloxan-induced diabetic rats by Elshater, Salman and Mussa (2009). This inhibitory effect caused by ginger may alse be due to the flavonoids that can be found in ginger rhizomes. There are different classes of flavonoids and according to Kwon, et al(2) a class of flavonoids called flavonols (specifically quercetin) are responsible for the inhibition of glucose transporter GLUT2 in the small intestine. Quercetin is the most abundant flavonol in ginger rhizome and may account for the results obtained.From the table, it can also be observed that the inhibitory effect of the two ginger varieties differs. The table shows that the yellow variety greatly inhibits glucose absorption with a rate of 0.67 mmol/L/min compared to common ginger, with a rate of 2.00 mmol/L/min. This difference may be accounted by the differential content of inhibitory substances found in both ginger varieties. Several studies had been conducted to compare the phenolic content of the two ginger varieties. In a study conducted by Danciu et al (2015), they claimed that there is an increase phenolic activity in the Curcuma longa variety of ginger (esp. in the rhizome) compared to the common one. This claim is supported by another study conducted by Alafiatayo (2014). Where they studied the phenolic activities of ten ginger varieties and found out that Curcuma longa and Zingiber officinale have the highest concentration of phenolic compounds. Among the two, Curcuma longa contains the highest concentration of flavonoids, phenolic acid and polyphenols. Statistical AnalysisFrom the values obtained by means of F-test using ANOVA, variations among the populations (4668.75) is much greater than the variations within the populations (418.75) and the calculated (11.15) is greater than (4.26), it can be concluded that at =0.05, there is at least one ginger variety that has a different glucose concentration.By calculating via pairwise means comparison test, it can be said that the mean glucose concentration between the control (83.75) and common (60) is not significant since there is a small interval between the two, and between control and yellow (20) is significant because of the huge difference in their respective means. It can also be deduced from the values obtained that the yellow ginger inhibits glucose absorption more efficiently than the common ginger as can be seen in its low mean glucose concentration.Errors that resulted in the inaccuracy of data involves the limitations of the Insight strips used wherein the optimal glucose concentration that it can detect is only up to 2000 mmol/L so glucose concentration higher than this value was not identified. One of the reasons also involves the unavailability of oxygen after the first tank was consumed during the determination of the glucose concentration in the small intestine.

SUMMARY AND CONCLUSIONThe effect of ginger extracts on the glucose absorption in rat small intestine was observed. Two ginger varieties, Zingiber officinale (common ginger) and Curcuma longa (yellow ginger), were compared in terms of their effect on the glucose absorption in rat small intestine. There were three experimental groups: the control group, given with normal rat food and water; the Zingiber officinale-treated group, which was given common ginger extracts via gavage method, and fed with normal rat food and water, and; the Curcuma longa-treated group, which was given yellow ginger extracts via gavage method, and fed with normal rat food and water. After two weeks of the experimental period, the rats were dissected. The glucose absorption in the rat small intestine was measured and determined by following a standard procedure. The initial and final glucose concentrations of all the rats small intestines were recorded. The average rates of glucose transport were also computed.Results showed that the control group had the highest rate of glucose transport, followed by the Z. officinale-treated group, then the C. longa-treated group. This could mean that the control group had the least glucose transport inhibition, and the C. longa-treated group had the most glucose transport inhibition. This may be due to the high phenolic compound and phenolic activity in the yellow ginger, which contributes to the inhibiting activity of gingers. From these results, it can be concluded that yellow ginger is more effective in inhibiting glucose transport in the small intestine of rats.Moreover, statistical analysis showed that there is a significant difference between the control group and the C. longa-treated group, and between the Z. officinale-treated group and the C. longa-treated group; no significant difference between the control group and the Zingiber officinale-treated group was observed. It is expected that the control group and the Z. officinale-treated group would have a significant difference, based on previous related studies. Human and mechanical errors and limitations of the apparatuses (e.g. Insight reagent strips) used may have contributed to this unexpected result. It is, therefore, recommended to use a reagent which gives a more detailed result and measure of the glucose concentration.

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